Enrichment of Marine Viruses via Size-Based Filtration and Centrifugal Concentration

摘要

Source: Heng, S. & Ceballos, R. M. Environmental Sampling of Photosynthetic Microbes and Their Viruses: From Field to Lab. J. Vis. Exp. (2025)

This video demonstrates the isolation and concentration of viruses from seawater using sequential filtration and centrifugal ultrafiltration. It outlines the steps for size-based viral separation and recovery of a concentrated suspension for downstream analysis.

方案

1. Field pre-processing of water samples
   NOTE: Upon arrival at an onshore facility (e.g., hotel room/conference room/field station), a series of pre-processing steps was undertaken to protect microbial content within the water samples and to separate different fractions of the seawater samples based on particle size.
   1. Filtration system assembly: Secure one end of a 1 m long piece of 9.525 mm inner diameter (I.D.) clear vinyl tubing to the hose barb inlet on the vacuum port of a portable air diaphragm vacuum pump. Secure the opposite end of the same hose to a 1.0 L, 0.45 µm filter vacuum cup assembly. Use both 0.45 µm and 0.22 µm filter cup assemblies in sequence to separate different fractions of the seawater sample as described below.
   2. To test the system for leaks or defective/torn filters, filter 0.25 L of seawater through the 0.45 µm polyethersulfone (PES) membrane filter cup. A non-leaky system will show suction between the filter and the grating once the seawater sample is completely filtered through.
   3. Filtration through 0.45 µm PES membrane filter cup assembly: To isolate photosynthetic microorganisms (e.g., microalgae and cyanobacteria), the goal is to filter 2 L of seawater sample through the 0.45 µm PES membrane filter cup using two 1 L filter cups (or four 500 mL filter cups). Appropriately label all filtrate bottles with filter size, depth from which the sample was taken, date, and sampling site designator. Finally, screw cap and cover bottles containing 0.45 µm filtrate with a semi-transparent cloth or sleeve to prevent excessive sunlight.
      NOTE: Do not allow the filter in the filter cup to run dry. Always keep a thin layer of liquid on the filter. The filter will accumulate biological debris (e.g., cells). If the filter becomes clogged with debris, carefully unscrew the cup assembly and set the used filter cup aside, but do not discard the clogged cup. Attach a new 0.45 µm filter and continue.
   4. Filtration through 0.22 µm PES membrane filter cup assembly: To isolate large eukaryotic viruses, use 1 L of the 0.45 µm filtrate from the previous step (step 1.3) and filter through a 0.22 µm filter cup. Appropriately label the ultrafiltrate bottle with filter size, depth from which the sample was taken, date, and sampling site designator. Finally, screw cap and cover the bottle containing 0.22 µm ultrafiltrate (from filtering the 0.45 µm filtrate).
      NOTE: Do not allow the filter in the filter cup to run dry. Always keep a thin layer of liquid on the filter. The filter will accumulate biological debris, which contains biological samples. If the filter becomes clogged, unscrew the cup assembly and set aside the used filter cup. Do not discard the clogged cup. Attach a new 0.22 µm filter and continue.
   5. Concentrate large virus biomass from 0.22 µm filter: Pour 14 mL of ultrafiltrate into each of two sterile 15 mL conical polypropylene centrifuge tubes. Use a sterile scalpel to cut along the outer rim of the used 0.22 µm filter within the cup assembly. Use sterile forceps to remove the biomass-laden and moist filter. Cut it in half. Roll up and insert one half of the filter paper into one of the 15 mL conical tubes. Repeat for the second half of the filter membrane and the second 15 mL conical tube. Screw on tube caps and secure with transparent film (e.g., Parafilm). Label each tube with filter size (i.e., 0.22 µm), site, depth, and date.
   6. Retain the remaining 0.45 µm filtrate as well as the remaining 0.22 µm filtrate for shipment back to the lab. Repeat this entire filtration process (steps 1.2 through 1.6) for each depth and sampling site.
      NOTE: Ultrafiltrate is used in the lab as a media base (with native minerals and other nutrients).

2. Sample processing to concentrate viruses/bacteriophage and enrich hosts
NOTE: This protocol describes the first processing steps required once samples reach the laboratory from the field site. Transport by airplane versus road vehicle may require different considerations. Environmental sample viability will decrease with abrupt changes in temperature, pH, salinity, exposure or lack of exposure to light, dissolved gas (e.g., carbon dioxide and oxygen) concentration, and time between environmental extraction and lab processing.

1. Spin concentrating and storing viruses and bacteriophages
   NOTE: Once samples arrive at the lab, processing within 8 h after arrival may minimize loss of viable microbes due to the time lapse between sample site extraction and arrival at the lab. The steps provided below will provide aliquots of concentrated bacteriophage (or virus) suspension (i.e., working stocks) for long-term storage and use.
   1. Concentrating ultrafiltrate: For each sampling depth by site, ship the remaining ~800 mL of ultrafiltrate to the lab. Use a 3K nominal molecular weight limit (NMWL) 15 mL centrifugal spin filter and centrifuge at 6000 x g for 10-20 min to concentrate 50-100 mL of ultrafiltrate down to 1.0-1.5 mL. Prepare grids for transmission electron microscopy (TEM) imaging to assess whether there are any virus-like particles (VLPs) in the resulting concentrated ultrafiltrate. If VLPs are detected, divide the concentrate into 100 mL aliquots. Store at 4 °C (or -20 °C for longer-term storage).
   2. Concentrating particles from 0.22 µm filter: Use a sterile scalpel to gently scrape debris from the 0.22 µm filters into the ultrafiltrate within the 50 mL tubes in which the filters were transported. Use a 10K NMWL spin-concentrator cup assembly to concentrate 50-200 mL of suspension down to a volume of ~3 mL of sample retentate. Prepare grids for TEM imaging to determine if there are VLPs from the 0.45 µm filtrate that may have been captured on the surface of the 0.22 µm filter. If VLPs are detected, divide the concentrate into 100 mL aliquots and store at 4 °C (or -20 °C for longer-term storage).

材料

本文使用的材料清单

姓名	公司	目录编号	评论
1.5 ml tubes	www.eppendorf.com	30123611
10K NMWL Centricon Plus 70 spin-concentrator	www.sigmaaldrich.com	UFC701008
3K NMWL Amicon 15 mL	www.sigmaaldrich.com	UFC800308
80ft long, 0.5” diameter, nylon	https://facomex.mx/producto/combo/	N/A
Centrifuge 5920 R	www.eppendorf.com	5948000107
Collapse-resistant rubber flex hose	swanhose.com	CELTF58050
Corning 50 mL centrifuge tubes	www.sigmaaldrich.com	CLS430291
Eppendorf tubes	www.sigmaaldrich.com	EP022364120
LVEM5 Benchtop Electron Microscope_Transmission electron microscopy (TEM)	delongamerica.com	N/A
Polyethersulfone (PES) membrane filter cup with 0.22 µm	www.sigmaaldrich.com	Z358193-1CS
Polyethersulfone (PES) membrane filter cup with 0.45 µm	www.sigmaaldrich.com	Z370622-1CS
Vacuum/pressure pump	Cole-Parmer; Vernon Hills, IL, USA	EW-79204-00
Vinyl tubing	www.homedepot.com	N/A